Qualitative and Quantitative Detection of Microbial Nucleic Acids

ABSTRACT

The present invention relates to new methods and uses for the qualitative and quantitative detection of microbial nucleic acids using at least a first control nucleic acid, or a first and a second control nucleic acid in different concentrations. The method is based on amplification of nucleic acids, for example the polymerase chain reaction. Further provided are kits comprising components for performing said methods and uses.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit of U.S. ProvisionalApplication No. 61/368,983 filed on Jul. 29, 2010. The entire disclosureof the above-referenced prior application is hereby incorporated hereinby reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to new methods and uses for thequalitative and quantitative detection of microbial nucleic acids usingat least a first control nucleic acid, or a first and a second controlnucleic acid in different concentrations. The method is based onamplification of nucleic acids, for example the polymerase chainreaction. Further provided are kits comprising components for performingsaid methods and uses.

BACKGROUND OF THE INVENTION

In the field of molecular diagnostics, the detection and quantificationof microbial nucleic acids using nucleic acid amplification reactionsplays a significant role. The routine screening of blood donations forthe presence of Hepatitis-C Virus (HCV), Human Immunodeficiency Virus(HIV), and/or Hepatitis-B Virus (HBV) is an example for the large-scaleapplication of nucleic acid amplification and detection reactions. Thelatter comprise a variety of different techniques, the most commonlyused one being the Polymerase Chain Reaction (PCR) introduced by KaryMullis in 1984. Automated systems for PCR-based analysis often make useof real-time detection of product amplification during the PCR process.Key to such methods is the use of modified oligonucleotides carryingreporter groups or labels.

Qualitative detection of a microbial nucleic acid in a biological sampleis crucial e.g. for recognizing an infection of an individual. Thereby,one important requirement for an assay for detection of a microbialinfection is that false-negative or false-positive results be avoided,since such results would almost inevitably lead to severe consequenceswith regard to treatment of the respective patient. Thus, especially inPCR-based methods, a qualitative internal control nucleic acid is addedto the detection mix. Said control is particularly important forconfirming the validity of a test result: At least in the case of anegative result with regard to the microbial nucleic acid, thequalitative internal control reaction has to perform reactive withingiven settings, i.e. the qualitative internal control must be detected,otherwise the test itself is considered to be inoperative. However, in aqualitative setup, said qualitative internal control does notnecessarily have to be detected in case of a positive result. Forqualitative tests, it is especially important that the sensitivity ofthe reaction is guaranteed and therefore strictly controlled As aconsequence, the concentration of the qualitative internal control mustbe relatively low so that even in a situation e.g. of slight inhibitionthe qualitative internal control is not be detected and therefore thetest is invalidated.

On the other hand and in addition to mere detection of the presence orabsence of a microbial nucleic acid in a sample, it is often importantto determine the quantity of said nucleic acid. As an example, stage andseverity of a viral disease may be assessed on the basis of the viralload. Further, monitoring of any therapy requires information on thequantity of a pathogen present in an individual in order to evaluate thetherapy's success. For a quantitative assay, it is necessary tointroduce a quantitative standard nucleic acid serving as a referencefor determining the absolute quantity of a microbial nucleic acid.Quantitation can be effectuated either by referencing to an externalcalibration or by implementing an internal quantitative standard.

In the case of an external calibration, standard curves are created inseparate reactions using known amounts of identical or comparablenucleic acids. The absolute quantity of a microbial nucleic acid issubsequently determined by comparison of the result obtained with theanalyzed sample with said standard function. External calibration,however, has the disadvantage that a possible extraction procedure, itsvaried efficacy, and the possible and often not predictable presence ofagents inhibiting the amplification and/or detection reaction are notreflected in the control.

This circumstance applies to any sample-related effects. Therefore, itmight be the case that a sample is judged as negative due to anunsuccessful extraction procedure or other sample-based factors, whereasthe microbial nucleic acid to be detected and quantified is actuallypresent in the sample.

For these and other reasons, an internal quantitative standard added tothe test reaction itself is of advantage. The internal quantitativestandard has at least the following two functions in a quantitativetest:

i) It monitors the validity of the reaction.

ii) It serves as reference in titer calculation thus compensating foreffects of inhibition and controlling the preparation and amplificationprocesses to allow a more accurate quantitation.

Therefore, in contrast to the qualitative internal control nucleic acidin a qualitative test which must be positive only in a target-negativereaction, the quantitative standard nucleic acid in a quantitative testhas two functions: reaction control and reaction calibration. Thereforeit must be positive and valid both in target-negative andtarget-positive reactions.

It further has to be suited to provide a reliable reference value forthe calculation of high nucleic acid concentrations. Thus, theconcentration of an internal quantitative standard nucleic acid needs tobe relatively high.

The qualitative internal control nucleic acid and/or the internalquantitative standard nucleic acid can be competitive, non-competitiveor partially competitive. A competitive qualitative internal controlnucleic acid and/or internal quantitative standard nucleic acid carriesessentially the same primer binding sites as the target and thuscompetes for the same primers as the target. Among the advantages of acompetitive setup is, e.g., that fewer sets of different primers have tobe introduced in the assay, thus reducing its costs and overallcomplexity. Furthermore, the functionality of the primers is monitoredas are inhibition effects which are target primer-specific. Anon-competitive qualitative internal control nucleic acid and/orinternal quantitative standard nucleic acid has different primer bindingsites than the target and thus binds to different primers. Advantages ofsuch a setup comprise, among others, the fact that the singleamplification events of the different nucleic acids in the reactionmixture can take place independently from each other without anycompetition effects. In a PCR using a partially competitive internalquantitative standard nucleic acid the respective control nucleic acidand at least one of the target nucleic acids compete for the sameprimers, while at least one other target nucleic acid binds to differentprimers.

Since the principles of a quantitative and a qualitative assay asdescribed above display different requirements when compared to eachother, also in view of regulatory requirements in various countries, thecommon approach used in the art has been the development of separatequantitative and qualitative assays for the same target nucleic acid,see e.g. Yang et al., J Agr Food Chem 2005, 53, 6222-6229.

The present invention provides an alternative solution displayingseveral advantages.

SUMMARY OF THE INVENTION

The present invention provides a method for simultaneously detecting andquantifying a microbial nucleic acid in a biological sample, the methodcomprising:

-   -   a) providing a reaction mixture comprising:        -   a first control nucleic acid,        -   one or more primer pairs that hybridize to distinct sequence            portions of the microbial nucleic acid and to distinct            sequence portions of the first control nucleic acid, and        -   two or more probes that hybridize to each of the sequences            amplified by the one or more primer pairs, wherein the            microbial nucleic acid and the first control nucleic acid            hybridize to different probes;    -   b) adding the biological sample to the reaction mixture;    -   c) performing one or more cycling steps, wherein each cycling        step comprises:        -   an amplifying step comprising producing one or more            amplification products derived from the microbial nucleic            acid if present in the sample and producing an amplification            product derived from the first control nucleic acid, and        -   a hybridizing step comprising hybridizing the amplification            products with the two or more probes, wherein the two or            more probes are each labeled with a donor fluorescent moiety            and a corresponding acceptor fluorescent moiety and each of            the two or more probes carries a different fluorescent            moiety dye; and    -   d) detecting and measuring fluorescent signals generated in step        c),

wherein the fluorescent signals generated by the first control nucleicacid and the microbial nucleic acid are proportional to theirconcentration and are indicative of the detection and quantification ofthe microbial nucleic acid, and wherein the fluorescent signals from thefirst control nucleic acid are indicative of an amplification occurringin the amplifying step even in the absence of the fluorescent signalsfrom the microbial nucleic acid.

In some embodiments, the fluorescent signals from the first controlnucleic acid are analyzed by different criteria to obtain a quantitativeresult and/or a qualitative result.

Additionally, the present invention provides a method for simultaneouslydetecting and quantifying a microbial nucleic acid in a biologicalsample, said method comprising:

-   -   a) providing a reaction mixture comprising:        -   a first and a second control nucleic acid in different            concentrations,        -   one or more primer pairs that hybridize to distinct sequence            portions of the microbial nucleic acid and to distinct            sequence portions of the first and the second control            nucleic acid, and        -   three or more probes that hybridize to each of the sequences            amplified by the one or more primer pairs, wherein the            microbial nucleic acid and the first and second control            nucleic acid each hybridize to different probes;    -   b) adding the biological sample to the reaction mixture;    -   c) performing one or more cycling steps, wherein each cycling        step comprises:        -   an amplifying step comprising producing one or more            amplification products derived from the microbial nucleic            acid if present in the sample and producing an amplification            product derived from the first control nucleic acid and an            amplification product derived from the second control            nucleic acid, and        -   a hybridizing step comprising hybridizing the amplification            products with the three or more probes, wherein the three or            more probes are each labeled with a donor fluorescent moiety            and a corresponding acceptor fluorescent moiety and each of            the three or more probes carries a different fluorescent            moiety dye; and    -   d) detecting and measuring fluorescent signals generated in step        c),

wherein the fluorescent signals generated by the first control nucleicacid and the microbial nucleic acid are proportional to theirconcentration and are indicative of the detection and quantification ofthe microbial nucleic acid, and wherein the fluorescent signals from thesecond control nucleic acid are indicative of an amplification occurringin the amplifying step even in the absence of the fluorescent signalsfrom the microbial nucleic acid.

In some embodiments, the first control nucleic acid is a quantitativestandard nucleic acid and the second control nucleic acid is aqualitative internal control nucleic acid. In other embodiments, themethods comprise in step d) determining the quantity of the microbialnucleic acid in the biological sample by comparison of the signalsgenerated by the microbial nucleic acid and the first control nucleicacid. A further embodiment employs a polymerase enzyme having 5′ to 3′exonuclease activity. In another embodiment of the invention, the firstcontrol nucleic acid is present in a concentration of 20-5000 times thelimit of detection of the microbial nucleic acid, and the second controlnucleic acid is present in a concentration of 1-25 times the limit ofdetection of the microbial nucleic acid. In another embodiment of theinvention, the first and second control nucleic acids are providedwithin one control reagent.

Additionally the present invention provides kits for simultaneouslydetecting and quantifying a microbial nucleic acid in a biologicalsample comprising a first and a second control nucleic acid in differentconcentrations, one or more primer pairs that hybridize to distinctsequence portions of the microbial nucleic acid and to distinct sequenceportions of the first and second control nucleic acid, and probes thathybridize to each of the sequences amplified by the one or more primerpairs, wherein the first and second control nucleic acids are providedwithin one control reagent.

The present invention further provides kits for simultaneously detectingand quantifying a microbial nucleic acid in a biological samplecomprising a first and a second control nucleic acid in differentconcentrations, one or more primer pairs that hybridize to distinctsequence portions of the microbial nucleic acid and to distinct sequenceportions of the first and second control nucleic acid, and probes thathybridize to each of the sequences amplified by the one or more primerpairs. In some embodiments, the first and second control nucleic acidare amplified by the same primer pair but hybridize to different probes.

Additionally the present invention provides an analytical system forsimultaneously detecting and quantifying a microbial nucleic acid in abiological sample comprising a sample preparation module comprising alysis buffer and a vessel for isolating and purifying the microbialnucleic acid, and an amplification and detection module comprising areaction receptacle in which the methods performed comprise:

-   -   a) providing a reaction mixture comprising:        -   a first control nucleic acid,        -   one or more primer pairs that hybridize to distinct sequence            portions of the microbial nucleic acid and to distinct            sequence portions of the first control nucleic acid, and        -   two or more probes that hybridize to each of the sequences            amplified by the one or more primer pairs, wherein the            microbial nucleic acid and the first control nucleic acid            hybridize to different probes;    -   b) adding the biological sample to the reaction mixture;    -   c) performing one or more cycling steps, wherein each cycling        step comprises:        -   an amplifying step comprising producing one or more            amplification products derived from the microbial nucleic            acid if present in the sample and producing an amplification            product derived from the first control nucleic acid, and        -   a hybridizing step comprising hybridizing the amplification            products with the two or more probes, wherein the two or            more probes are each labeled with a donor fluorescent moiety            and a corresponding acceptor fluorescent moiety and each of            the two or more probes carries a different fluorescent            moiety dye; and    -   d) detecting and measuring fluorescent signals generated in step        c),

wherein the fluorescent signals generated by the first control nucleicacid and the microbial nucleic acid are proportional to theirconcentration and are indicative of the detection and quantification ofthe microbial nucleic acid, and wherein the fluorescent signals from thefirst control nucleic acid are indicative of an amplification occurringin the amplifying step even in the absence of the fluorescent signalsfrom the microbial nucleic acid.

Further, the amplification and detection module of the invention canfurther comprise a reaction receptacle in which the methods performedcomprise:

-   -   a) providing a reaction mixture comprising:        -   a first and a second control nucleic acid in different            concentrations,        -   one or more primer pairs that hybridize to distinct sequence            portions of the microbial nucleic acid and to distinct            sequence portions of the first and the second control            nucleic acid, and        -   three or more probes that hybridize to each of the sequences            amplified by the one or more primer pairs, wherein the            microbial nucleic acid and the first and second control            nucleic acid each hybridize to different probes;    -   b) adding the biological sample to the reaction mixture;    -   c) performing one or more cycling steps, wherein each cycling        step comprises:        -   an amplifying step comprising producing one or more            amplification products derived from the microbial nucleic            acid if present in the sample and producing an amplification            product derived from the first control nucleic acid and an            amplification product derived from the second control            nucleic acid, and        -   a hybridizing step comprising hybridizing the amplification            products with the three or more probes, wherein the three or            more probes are each labeled with a donor fluorescent moiety            and a corresponding acceptor fluorescent moiety and each of            the three or more probes carries a different fluorescent            moiety dye; and    -   d) detecting and measuring fluorescent signals generated in step        c),

wherein the fluorescent signals generated by the first control nucleicacid and the microbial nucleic acid are proportional to theirconcentration and are indicative of the detection and quantification ofthe microbial nucleic acid, and wherein the fluorescent signals from thesecond control nucleic acid are indicative of an amplification occurringin the amplifying step even in the absence of the fluorescent signalsfrom the microbial nucleic acid.

The present invention further provides an analytical system additionallycomprising a transfer module for transferring the biological sample fromthe sample preparation module to the reaction receptacle. In someembodiments, the first control nucleic acid is a quantitative standardnucleic acid and the second control nucleic acid is a qualitativeinternal control nucleic acid. In other embodiments, the methodscomprise in step d) determining the quantity of the microbial nucleicacid in the biological sample by comparison of the signals generated bythe microbial nucleic acid and the first control nucleic acid. A furtherembodiment employs a polymerase enzyme having 5′ to 3′ exonucleaseactivity. In another embodiment of the invention, the first controlnucleic acid is present in a concentration of 20-5000 times the limit ofdetection of the microbial nucleic acid, and the second control nucleicacid is present in a concentration of 1-25 times the limit of detectionof the microbial nucleic acid. In another embodiment of the invention,the first and second control nucleic acids are provided within onecontrol reagent.

DESCRIPTION OF THE INVENTION

The present invention relates to new methods and uses for thesimultaneous qualitative and quantitative detection of microbial nucleicacids. The method is based on amplification of nucleic acids, forexample the polymerase chain reaction. In brief, specific sequenceportions of the microbial nucleic acid and at least a first controlnucleic acid are amplified and detected using one or more specificprimer pairs. Thus, one subject of the invention is:

A method for simultaneously detecting and quantifying a microbialnucleic acid in a biological sample, said method comprising:

a) isolating and purifying said microbial nucleic acid

b) providing a reaction mixture comprising at least a first controlnucleic acid, one or more primer pairs specifically hybridizing todistinct sequence portions of said microbial nucleic acid and todistinct sequence portions of said control nucleic acid, and probesspecifically hybridizing to each of the sequences amplified by said oneor more primer pairs, wherein said microbial nucleic acid and saidcontrol nucleic acid hybridize to different probes

c) adding said biological sample to said reaction mixture

d) performing one or more cycling steps, wherein a cycling stepcomprises an amplifying step, said amplifying step comprising producingone or more amplification products derived from said microbial nucleicacid if present in said sample and producing an amplification productderived from said control nucleic acid, and wherein a cycling stepcomprises a hybridizing step, said hybridizing step comprisinghybridizing the sequences amplified by said primer pair with saidprobes, wherein the probes are labeled with a donor fluorescent moietyand a corresponding acceptor fluorescent moiety and each of the probescarries a different fluorescent dye

e) detecting and measuring fluorescent signals generated by saidamplification products and being proportional to the concentration ofsaid control nucleic acid and said microbial nucleic acid, wherein thepresence of an amplification product of said control nucleic acid isindicative of an amplification occurring in the reaction mixture even inthe absence of an amplification product for said microbial nucleic acid.

The present invention, for example, allows for a common design ofqualitative and quantitative assays for any given microbial nucleic acidusing identical reagents.

In some embodiments, the first control nucleic acid described above mayserve as both an internal quantitative standard nucleic acid and aqualitative internal control nucleic acid. Thus, it abolishes the needfor two separate tests or test kits, respectively. The performance oftwo separate tests puts a burden on the patient due to additional blooddraws and potentially imposes additional costs on the healthcare systemsin case the quantitative and the qualitative test are both reimbursed.Furthermore, the time-to-result for both a qualitative and aquantitative test is reduced when compared to sequential qualitative andquantitative experiments.

In some embodiments, said first control nucleic acid is evaluatedaccording to different criteria for obtaining a qualitative and aquantitative result. In one embodiment, the same set of raw dataobtained during real-time PCR is analyzed against two different testparameter settings. The qualitative test parameter settings applied fora valid qualitative control nucleic acid are more stringent as comparedto the parameter settings applied for the internal quantitative standardneeded for a quantitative result output. The settings for the internalquantitative standard nucleic acid in a quantitative reaction cannot beas stringent as the presence of target may affect the respective growthcurves of the internal quantitative standard nucleic acid (see FIG. 1a).

Thus, another embodiment of the invention is the method described above,wherein the first control nucleic acid is analyzed according todifferent criteria in order to serve as a quantitative standard nucleicacid or as a qualitative internal control nucleic acid.

In other embodiments, it can be advantageous to employ a first and asecond control nucleic acid in different concentrations due to thefollowing reasons: Usually, the internal quantitative standard nucleicacid or acids in quantitative tests have a rather high concentration sothat they are still amplified and detected in samples with a highconcentration of target nucleic acid. Therefore, its usability as acontrol for the detection of low positive samples close to the limit ofdetection (LOD) is sometimes not given, especially if the amplificationreaction is partly suppressed. In that case, the detection of a highlyconcentrated control nucleic acid cannot serve as a measure ofsufficient sensitivity of the respective assay.

Thus, another aspect of the present invention is the following:

A method for simultaneously detecting and quantifying a microbialnucleic acid in a biological sample, said method comprising:

a) isolating and purifying said microbial nucleic acid

b) providing a reaction mixture comprising a first and a second controlnucleic acid in different concentrations, one or more primer pairsspecifically hybridizing to distinct sequence portions of said microbialnucleic acid and to distinct sequence portions of said control nucleicacids, and probes specifically hybridizing to each of the sequencesamplified by said one or more primer pairs, wherein said microbialnucleic acid and said first control nucleic acid and said second controlnucleic acid hybridize to different probes

c) adding said biological sample to said reaction mixture

d) performing one or more cycling steps, wherein a cycling stepcomprises an amplifying step, said amplifying step comprising producingone or more amplification products derived from said microbial nucleicacid if present in said sample and producing an amplification productderived from said first control nucleic acid and said second controlnucleic acid, and wherein a cycling step comprises a hybridizing step,said hybridizing step comprising hybridizing the sequences amplified bysaid primer pair with said probes, wherein the probes are labeled with adonor fluorescent moiety and a corresponding acceptor fluorescent moietyand each of the probes carries a different fluorescent dye

e) detecting and measuring fluorescent signals generated by theamplification products of said first control nucleic acid and saidmicrobial nucleic acid and being proportional to their concentration,and/or simultaneously detecting fluorescent signals generated by saidamplification product of said second control nucleic acid, wherein thepresence of an amplification product of said second control nucleic acidis indicative of an amplification occurring in the reaction mixture evenin the absence of an amplification product for said microbial nucleicacid.

The exploitation of a first and a second control nucleic acid indifferent concentrations within the same control reagent, according tothe present invention, overcomes the above-stated problem. The nucleicacid with the lower or lowest concentration corresponds to a qualitativeinternal control nucleic acid for a qualitative assay and ensures theability to judge whether a negative result is valid or not, i.e. anegative result is not valid in case the internal control nucleic acidis not detected.

According to the present invention, the artisan can take advantage ofthe synergies between developing a quantitative and a qualitative setupby performing both in a single assay, thus reducing development costs,requiring less material and working force, reducing manufacturing costsand also cutting down the required time to establish an assay.Furthermore, double testing of patients putting burden on the patientdue to additional blood draws is avoided as well as costs for the healthcare systems, where quantitative and qualitative test are bothreimbursed, are reduced.

The qualitative internal control nucleic acid and/or the internalquantitative standard nucleic acid can be competitive, non-competitiveor partially competitive.

“Competitive” means that, in an amplification reaction comprisingprimers, the respective control nucleic acid and the target nucleic acidor acids have at least essentially the same primer binding sites andthus compete for the same primers. Among the advantages of a competitivesetup is, e.g., that fewer sets of different primers have to beintroduced in the assay, thus reducing its costs and overall complexity.Furthermore, the functionality of the primers is monitored as areinhibition effects which are target primer specific.

“Non-competitive” means that the respective control nucleic acid and thetarget nucleic acid or acids have different primer binding sites andthus bind to different primers. Advantages of such a setup comprise,among others, the fact that the single amplification events of thedifferent nucleic acids in the reaction mixture can take placeindependently from each other without any competition effects.

“Partially competitive” means that the respective control nucleic acidand at least one of the target nucleic acids compete for the sameprimers, while at least one other target nucleic acid binds to differentprimers.

By using an internal quantitative standard nucleic acid as a firstcontrol nucleic acid according to the method or methods of theinvention, sample-specific, but also sample-unspecific inhibitoryeffects possibly interfering with the amplification and detectionreactions for quantitative standard nucleic acid and microbial nucleicacid simultaneously (target region-independent inhibition) are leveledresulting in more accurate titers. “Internal” means that the firstcontrol nucleic acid is amplified, detected and quantified within thesame reaction mixture as the microbial nucleic acid instead of in aseparate experiment.

Another aspect of the invention is the method described supra, whereinthe first control nucleic acid is a quantitative standard nucleic acidand the second control nucleic acid is a qualitative internal controlnucleic acid.

The person skilled in the art can extract reliable qualitative, butoptionally also equally reliable quantitative information from the sameexperiment. Therefore, in another aspect the invention relates to thefollowing:

The method described above, further comprising

in step e) determining the quantity of said microbial nucleic acid insaid biological sample by comparison of the signals generated by saidmicrobial nucleic acid and said first control nucleic acid.

In another embodiment, the first control nucleic acid and the secondcontrol nucleic acid have essentially the same sequence. In anotherembodiment, they have the same primer binding sites, but different probebinding sites.

This bears the advantage that the control nucleic acids for qualitativeand quantitative measurements can based on the same selected bindingsequence and share the same advantageous properties with respect toassay performance and in view of the analyte sequence or sequences.

Conventional techniques of molecular biology and nucleic acid chemistry,which are within the skill of the art, are explained in the literature.See, for example, Sambrook J. et al., Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1989, Gait, M. J., ed., 1984; Nucleic Acid Hybridization, Hames, B. D.,and Higgins, S. J., eds., 1984; and a series, Methods in Enzymology,Academic Press, Inc.

“Simultaneously”, in the sense of the invention, means that two actions,such as e.g. detecting and quantifying of a nucleic acid, are performedwithin the same reaction or reaction mixture.

A “reaction mixture” as used in the present invention comprises at leastall components to facilitate a biological or chemical reaction. It is asingle volume without any separating compartments, i.e. all componentspresent in said “reaction mixture” are in immediate contact with eachother.

A “biological sample” can be any sample of natural origin. An example ofa “biological sample” is derived from a human and is a body liquid. Inan embodiment of the invention, the “biological sample” is blood.

As is known in the art, a “nucleoside” is a base-sugar combination. Thebase portion of the nucleoside is normally a heterocyclic base. The twomost common classes of such heterocyclic bases are the purines and thepyrimidines.

“Nucleotides” are “nucleosides” that further include a phosphate groupcovalently linked to the sugar portion of the nucleoside. For those“nucleosides” that include a pentofuranosyl sugar, the phosphate groupcan be linked to either the 2′, 3′ or 5′ hydroxyl moiety of the sugar. A“nucleotide” is the “monomeric unit” of an “oligonucleotide”, moregenerally denoted herein as an “oligomeric compound”, or a“polynucleotide”, more generally denoted as a “polymeric compound”.Another general expression for the aforementioned is deoxyribonucleicacid (DNA) and ribonucleic acid (RNA).

According to the invention, an “oligomeric compound” is a compoundconsisting of “monomeric units” which may be “nucleotides” alone or“non-natural compounds” (see below), more specifically “modifiednucleotides” (or “nucleotide analogs”) or “non-nucleotide compounds”,alone or combinations thereof. “Oligonucleotides” and “modifiedoligonucleotides” (or “oligonucleotide analogs”) are subgroups of“oligomeric compounds” in the context of the invention.

In the context of this invention, the term “oligonucleotide” refers to“polynucleotides” formed from a plurality of “nucleotides” as the“monomeric unit”, i.e. an “oligonucleotide” belongs to a specificsubgroup of an “oligomeric compound” or “polymeric compound” ofribonucleic acid (RNA) or deoxyribonucleic acid (DNA) with “monomericunits”. The phosphate groups are commonly referred to as forming theinternucleoside backbone of the “oligonucleotide”. The normal linkage orbackbone of RNA and DNA is a 3′ to 5′ phosphodiester linkage.

“Oligonucleotides” and “modified oligonucleotides” (see below) accordingto the invention may be synthesized as principally described in the artand known to the expert in the field. Methods for preparing oligomericcompounds of specific sequences are known in the art, and include, forexample, cloning and restriction of appropriate sequences and directchemical synthesis. Chemical synthesis methods may include, for example,the phosphotriester method described by Narang S. A. et al., Methods inEnzymology 68 (1979) 90-98, the phosphodiester method disclosed by BrownE. L., et al., Methods in Enzymology 68 (1979) 109-151, thephosphoramidite method disclosed in Beaucage et al., Tetrahedron Letters22 (1981) 1859, the H-phosphonate method disclosed in Garegg et al.,Chem. Scr. 25 (1985) 280-282 and the solid support method disclosed inU.S. Pat. No. 4,458,066.

For the above-described method, the nucleic acids can be present indouble-stranded or single-stranded form whereby the double-strandednucleic acids are denatured, i.e. made single-stranded, before themethod is performed by heating, i.e. thermal denaturing.

In another embodiment, a primer and/or the probe may be chemicallymodified, i.e. the primer and/or the probe comprise a modifiednucleotide or a non-nucleotide compound. The probe or the primer is thena modified oligonucleotide.

“Modified nucleotides” (or “nucleotide analogs”) differ from a natural“nucleotide” by some modification but still consist of a base, apentofuranosyl sugar, a phosphate portion, base-like, pentofuranosylsugar-like and phosphate-like portion or combinations thereof. Forexample, a “label” may be attached to the base portion of a “nucleotide”whereby a “modified nucleotide” is obtained. A natural base in a“nucleotide” may also be replaced by e.g. a 7-deazapurine whereby a“modified nucleotide” is obtained as well. The terms “modifiednucleotide” or “nucleotide analog” are used interchangeably in thepresent application. A “modified nucleoside” (or “nucleoside analog”)differs from a natural nucleoside by some modification in the manner asoutlined above for a “modified nucleotide” (or a “nucleotide analog”).

A “non-nucleotide compound” is different from a natural “nucleotide” butis in the sense of this invention still capable—similar to a“nucleotide”—of being a “monomeric unit” of an “oligomeric compound”.Therefore, a “non-nucleotide compound” has to be capable of forming an“oligomeric compound” with “nucleotides”. Even “non-nucleotidecompounds” may contain base-like, pentofuranosyl sugar-like orphosphate-like portions, however, not all of them are present at thesame time in a “non-nucleotide compound”.

A “modified oligonucleotide” (or “oligonucleotide analog”), belonging toanother specific subgroup of the “oligomeric compounds”, possesses oneor more “nucleotides”, one or more “non-nucleotide compounds” or“modified nucleotides” as “monomeric units”. Thus, the term “modifiedoligonucleotide” (or “oligonucleotide analog”) refers to structures thatfunction in a manner substantially similar to “oligonucleotides” and isused interchangeably throughout the application. From a synthetic pointof view, a “modified oligonucleotide” (or a “oligonucleotide analog”)can for example be made by chemical modification of “oligonucleotides”by appropriate modification of the phosphate backbone, ribose unit orthe nucleotide bases (Uhlmann and Peyman, Chemical Reviews 90 (1990)543; Verma S., and Eckstein F., Annu. Rev. Biochem. 67 (1998) 99-134).Representative modifications include phosphorothioate,phosphorodithioate, methyl phosphonate, phosphotriester orphosphoramidate inter-nucleoside linkages in place of phosphodiesterinter-nucleoside linkages; deaza- or aza-purines and -pyrimidines inplace of natural purine and pyrimidine bases, pyrimidine bases havingsubstituent groups at the 5 or 6 position; purine bases having alteredsubstituent groups at the 2, 6 or 8 positions or 7 position as7-deazapurines; bases carrying alkyl-, alkenyl-, alkinyl oraryl-moieties, e.g. lower alkyl groups such as methyl, ethyl, propyl,butyl, tert-butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, or arylgroups like phenyl, benzyl, naphtyl; sugars having substituent groupsat, for example, their 2′ position; or carbocyclic or acyclic sugaranalogs. Other modifications consistent with the spirit of thisinvention are known to those skilled in the art. Such “modifiedoligonucleotides” (or “oligonucleotide analogs”) are best described asbeing functionally interchangeable with, yet structurally differentfrom, natural “oligonucleotides” (or synthetic “oligonucleotides” alongnatural lines). In more detail, exemplary modifications are disclosed inVerma S., and Eckstein F., Annu. Rev. Biochem. 67 (1998) 99-134 or WO02/12263. In addition, modification can be made wherein nucleoside unitsare joined through groups that substitute for the internucleosidephosphate or sugar phosphate linkages. Such linkages include thosedisclosed in Verma S., and Eckstein F., Annu. Rev. Biochem. 67 (1998)99-134. When other than phosphate linkages are utilized to link thenucleoside units, such structures have also been described as“oligonucleosides”.

A “nucleic acid” as well as the “target nucleic acid” or the “microbialnucleic acid” is a polymeric compound of “nucleotides” as known to theexpert skilled in the art. “Target nucleic acid” or “microbial nucleicacid” is used herein to denote a “nucleic acid” in a sample which shouldbe analyzed, i.e. the presence, non-presence and/or amount thereof in asample should be determined. Therefore, in this case the nucleic acid isthe target and can therefore be also denoted as “target nucleic acid”.Since, according to the invention, the target nucleic acid is ofmicrobial origin, the target nucleic acid is also referred to as“microbial nucleic acid”. For example, if it has to be determinedwhether blood contains HCV, the “target nucleic acid” or “microbialnucleic acid” is the nucleic acid of HCV.

“Microorganism” means any virus, bacterium, archaean, fungus or anyunicellular eukaryotic organism.

“Microbial” means derived from or belonging to a “microorganism”.

“Detecting” means determining the presence or absence of a specificobject, such as a target, or signal.

“Quantifying” means determining the quantity of a specific object, suchas a target, or signal.

“Measuring” means determining at least a relative value of a specificobject, such as a target, or signal.

In the sense of certain embodiments of the invention using a first and asecond control nucleic acid, the first control nucleic acid serves as an“internal quantitative standard nucleic acid”. An “internal quantitativestandard nucleic acid” is a “nucleic acid” and thus a polymeric compoundof “nucleotides” as known to the expert skilled in the art. In the caseof the “internal quantitative standard nucleic acid”, the nucleic acidis apt to be and used simultaneously as control for the validity of thereaction and as a reference in order to “quantify”, i.e. to determinethe quantity of the “target nucleic acid” or “microbial nucleic acid”.For this purpose, the “internal quantitative standard nucleic acid”undergoes all possible sample preparation steps along with the “targetnucleic acid” or the “microbial nucleic acid”. Moreover, it is processedthroughout the method within the same reaction mixture. The “internalquantitative standard nucleic acid” must generate, directly orindirectly, a detectable signal both in the presence or absence of thetarget nucleic acid. For this purpose, the concentration of the“internal quantitative standard nucleic acid” has to be carefullyoptimized in each test in order not to interfere with sensitivity but inorder to generate a detectable signal also e.g. at very high targetconcentrations. For example, the concentration range for the “internalquantitative standard nucleic acid”, i.e. the first control nucleicacid, will comprise a range of 100 copies per reaction to 100 000 copiesper reaction. Possible concentrations of the “internal quantitativestandard nucleic acid” may be e.g. for HIV: 1000 copies/reaction, forHCV: 7500 copies/reaction, but these concentrations may be adapted tothe specific assay as known by the person skilled in the art. In termsof the limit of detection (LOD) of the respective assay, theconcentration range for the “internal quantitative standard nucleicacid” is for example 20-5000×LOD, or 20-1000×LOD, or 20-500×LOD. Thefinal concentration of the “internal quantitative standard nucleic acid”in the reaction mixture is dependent on the quantitative measuring rangeaccomplished. The “internal quantitative standard nucleic acid” can be,for example, DNA, RNA or PNA, armored DNA or armored RNA and modifiedforms thereof.

Further, in the sense of the invention, the second control nucleic acidserves as a “qualitative internal control nucleic acid”. A “qualitativeinternal control nucleic acid” is particularly important for confirmingthe validity of the test result of a qualitative detection assay: Atleast in the case of a negative result, the qualitative internal controlnucleic acid must be detected, otherwise the test itself is consideredto be inoperative. However, in a qualitative setup, the qualitativeinternal control nucleic acid does not necessarily have to be detectedin case of a positive result. For qualitative tests, it is importantthat the sensitivity of the reaction is guaranteed and thus strictlycontrolled. As a consequence, the concentration of the qualitativeinternal control nucleic acid must be relatively low, such that even ina situation of slight inhibition the test is considered invalid. It hasto be carefully adapted to the respective assay and its sensitivity. Forexample, the concentration range for the “qualitative internal nucleicacid”, i.e. the second control nucleic acid, comprises a range of 1 copyper reaction to 1000 copies per reaction. In relation to the respectiveassay's limit of detection (LOD), its concentration is for examplebetween the LOD of an assay and the 25fold value of the LOD. In anotherexample, it is between 2× and 20×LOD, or between 2× and 15×LOD, orbetween 2× and 10×LOD. In another embodiment, it is between 3× and7×LOD.

“Limit of detection” or “LOD” means the lowest detectable amount orconcentration of a nucleic acid in a sample with a predefined hitrate. Alow “LOD” corresponds to high sensitivity and vice versa. The “LOD” isusually expressed either by means of the unit “cp/ml”, particularly ifthe nucleic acid is a viral nucleic acid, or as IU/ml. “Cp/ml” means“copies per milliliter” wherein a “copy” is copy of the respectivenucleic acid. IU/ml stands for “International units/ml”, referring tothe WHO standard. Depending on the target, LOD values in clinicalmolecular diagnostic assays are typically below 1000 cp/ml. In example,the LOD in assays performed in the context of the invention is between 1and 500 cp/ml.

A widely used method for calculating an LOD is “Probit Analysis”, whichis a method of analyzing the relationship between a stimulus (dose) andthe quantal (all or nothing) response. In a typical quantal responseexperiment, groups of animals are given different doses of a drug. Thepercent dying at each dose level is recorded. These data may then beanalyzed using Probit Analysis. The Probit Model assumes that thepercent response is related to the log dose as the cumulative normaldistribution. That is, the log doses may be used as variables to readthe percent dying from the cumulative normal. Using the normaldistribution, rather than other probability distributions, influencesthe predicted response rate at the high and low ends of possible doses,but has little influence near the middle.

The “Probit Analysis” can be applied at distinct “hitrates”. As known inthe art, “hitrate” is commonly expressed in percent [%] and indicatesthe percentage of positive results at a specific concentration of ananalyte. Thus for example, an LOD can be determined at 95% hitrate,which means that the LOD is calculated for a setting in which 95% of thevalid results are positive.

The term “primer” is used herein as known to the expert skilled in theart and refers to “oligomeric compounds”, primarily to“oligonucleotides”, but also to “modified oligonucleotides” that areable to “prime” DNA synthesis by a template-dependent DNA polymerase,i.e. the 3′-end of the e.g. oligonucleotide provides a free 3′-OH groupwhereto further “nucleotides” may be attached by a template-dependentDNA polymerase establishing 3′ to 5′ phosphodiester linkage wherebydeoxynucleoside triphosphates are used and whereby pyrophosphate isreleased.

The term “probe”, in the context of the invention, is also anoligonucleotide, but with a specific function: It hybridizes to othernucleic acids in a reaction mixture, in order to enable their detection.Thus, for example the probes specifically bind to certain nucleic acids.A probe carries, for example, at least one label. Probes can e.g. belabeled with different dyes, such that the respective probes can bedetected and measured independently from each other.

“Labels”, often referred to as “reporter groups”, are generally groupsthat make a nucleic acid, in particular the “oligomeric compound” or the“modified oligonucleotide”, as well as any nucleic acids bound theretodistinguishable from the remainder of the sample (nucleic acids havingattached a “label” can also be termed labeled nucleic acid bindingcompounds, labeled probes or just probes). Labels according to theinvention can include for example fluorescent labels, which are e.g.“fluorescent dyes” as a fluorescein dye, a rhodamine dye, a cyanine dye,and a coumarin dye. Exemplary “fluorescent dyes” according to theinvention are FAM, HEX, CY5, JA270, Cyan, CY5.5, LC-Red 640, LC-Red 705.

For the above-described method, the nucleic acids can be present indouble-stranded or single-stranded form whereby the double-strandednucleic acids are denatured, i.e. made single-stranded, before themethod is performed by heating, i.e. thermal denaturing.

In another embodiment, a primer and/or the probe may be chemicallymodified, i.e. the primer and/or the probe comprise a modifiednucleotide or a non-nucleotide compound. The probe or the primer is thena modified oligonucleotide.

A “detectable signal” is a signal “generated”, by a compound such as the“microbial nucleic acid”, the “internal control nucleic acid” or the“quantitative standard nucleic acid”, rendering said compounddistinguishable from the remainder of the sample. According to theinvention, said “detectable signal” can be quantified or analyzed in aqualitative manner. A “detectable signal” can be e.g. radioactive oroptical such as luminescent signals. For example, “detectable signals”according to the invention can be fluorescent signals emitted by“fluorescent dyes”.

“Inhibition” or “suppression” of a PCR reaction denotes a PCR reactionwhich is less efficient as compared to the standard reaction observed inthe majority of samples. The “inhibition” or “suppression” effect can beseen in either reduced fluorescence levels of growth curves for controland/or target, in delayed CT values, in changes of the slope of thegrowth curve, in changes in the turning point or other features of thereaction characteristics. The “inhibition” or “suppression” effect canresult from varied sample preparation efficacy, sample-related effects,the possible and often not predictable presence of agents inhibiting theamplification and/or detection reaction, and other reasons. Therefore,it might be the case that a sample is judged as negative due to anunsuccessful extraction procedure or other sample-based factors, whereasthe microbial nucleic acid to be detected and quantified is actuallypresent in the sample.

“To generate” means to produce, directly or indirectly. In the contextof a “detectable signal”, “to generate” can therefore mean “to producedirectly”, e.g. in the case of a fluorescent dye emitting a fluorescentsignal, or “to produce indirectly” in the sense of “to evoke” or “toinduce”, such as a “microbial nucleic acid” “generating” a “detectablesignal” via a “label” such as a “fluorescent dye”, or via a nucleic acidprobe carrying a “label” such as a “fluorescent dye”.

As known by the person skilled in the art, the terms “specific” or“specifically hybridizing” in the context of primers and probes impliesthat a primer or probe “specific” for a distinct nucleic acid binds tosaid nucleic acid under stringent conditions. For example, the primersand probes used in the method according to the invention are at least80% identical to sequence portions of the microbial nucleic acid and/orthe first and second control nucleic acid.

The “Polymerase Chain Reaction” (PCR) is disclosed, among otherreferences, in U.S. Pat. Nos. 4,683,202, 4,683,195, 4,800,159, and4,965,188 and is an exemplary nucleic acid amplification technique usedfor method according to the invention. PCR typically employs two or moreoligonucleotide primers that bind to a selected nucleic acid template(e.g. DNA or RNA). Primers useful in the present invention includeoligonucleotides capable of acting as a point of initiation of nucleicacid synthesis within the nucleic acid sequences of the microbialnucleic acid or quantitative standard nucleic acid. A primer can bepurified from a restriction digest by conventional methods, or it can beproduced synthetically. The primer is for example single-stranded formaximum efficiency in amplification, but the primer can bedouble-stranded. Double-stranded primers are first denatured, i.e.,treated to separate the strands. One method of denaturing doublestranded nucleic acids is by heating. A “thermostable polymerase” is apolymerase enzyme that is heat stable, i.e., it is an enzyme thatcatalyzes the formation of primer extension products complementary to atemplate and does not irreversibly denature when subjected to theelevated temperatures for the time necessary to effect denaturation ofdouble-stranded template nucleic acids. Generally, the synthesis isinitiated at the 3′ end of each primer and proceeds in the 5′ to 3′direction along the template strand. Thermostable polymerases have beenisolated from Thermus flavus, T. ruber, T. thermophilus, T. aquaticus,T. lacteus, T. rubens, Bacillus stearothermophilus, and Methanothermusfervidus. Nonetheless, polymerases that are not thermostable also can beemployed in PCR assays provided the enzyme is replenished. If thetemplate nucleic acid is double-stranded, it is necessary to separatethe two strands before it can be used as a template in PCR. Strandseparation can be accomplished by any suitable denaturing methodincluding physical, chemical or enzymatic means. One method ofseparating the nucleic acid strands involves heating the nucleic aciduntil it is predominately denatured (e.g., greater than 50%, 60%, 70%,80%, 90% or 95% denatured). The heating conditions necessary fordenaturing template nucleic acid will depend, e.g., on the buffer saltconcentration and the length and nucleotide composition of the nucleicacids being denatured, but typically range from about 90° C. to about105° C. for a time depending on features of the reaction such astemperature and the nucleic acid length. Denaturation is typicallyperformed for about 3 sec to 4 min (e.g., 5 sec to 2 min 30 sec, or 10sec to 1.5 min). If the double-stranded template nucleic acid isdenatured by heat, the reaction mixture is allowed to cool to atemperature that promotes annealing of each primer to its targetsequence on the microbial nucleic acid and/or internal standard nucleicacid. The temperature for annealing is usually from about 35° C. toabout 75° C. (e.g., about 40° C. to about 70° C.; about 45° C. to about66° C.). Annealing times can be from about 5 sec to about 1 min (e.g.,about 10 sec to about 50 sec; about 15 sec to about 40 sec The reactionmixture is then adjusted to a temperature at which the activity of thepolymerase is promoted or optimized, i.e., a temperature sufficient forextension to occur from the annealed primer to generate productscomplementary to the microbial nucleic acid and/or internal standardnucleic acid. The temperature should be sufficient to synthesize anextension product from each primer that is annealed to a nucleic acidtemplate, but should not be so high as to denature an extension productfrom its complementary template (e.g., the temperature for extensiongenerally ranges from about 35° to 80° C. (e.g., about 40° C. to about75° C.; about 45° C. to 72° C.). Extension times can be from about 5 secto about 5 min (e.g., about 10 sec to about 3 min; about 15 sec to about2 min; about 20 sec to about 1 min). The newly synthesized strands forma double-stranded molecule that can be used in the succeeding steps ofthe reaction. The steps of strand separation, annealing, and elongationcan be repeated as often as needed to produce the desired quantity ofamplification products corresponding to the microbial nucleic acidand/or quantitative standard nucleic acid. The limiting factors in thereaction are the amounts of primers, thermostable enzyme, and nucleosidetriphosphates present in the reaction. The cycling steps (i.e.,denaturation, annealing, and extension) are repeated at least once. Foruse in detection, the number of cycling steps will depend, e.g., on thenature of the sample. If the sample is a complex mixture of nucleicacids, more cycling steps will be required to amplify the targetsequence sufficient for detection. Generally, the cycling steps arerepeated at least about 20 times, but may be repeated as many as 40, 60,or even 100 times.

Nucleic acid amplification reactions apart from PCR comprise the LigaseChain Reaction (LCR; Wu D. Y. and Wallace R. B., Genomics 4 (1989)560-69; and Barany F., Proc. Natl. Acad. Sci. USA 88 (1991) 189-193);Polymerase Ligase Chain Reaction (Barany F., PCR Methods and Applic. 1(1991) 5-16); Gap-LCR (WO 90/01069); Repair Chain Reaction (EP 0439182A2), 3SR (Kwoh D. Y. et al., Proc. Natl. Acad. Sci. USA 86 (1989)1173-1177; Guatelli J. C., et al., Proc. Natl. Acad. Sci. USA 87 (1990)1874-1878; WO 92/08808), and NASBA (U.S. Pat. No. 5,130,238). Further,there are strand displacement amplification (SDA), transcriptionmediated amplification (TMA), and Q-beta-amplification (for a review seee.g. Whelen A. C. and Persing D. H., Annu Rev. Microbiol. 50 (1996)349-373; Abramson R. D. and Myers T. W., Curr Opin Biotechnol 4 (1993)41-47).

Suitable nucleic acid detection methods are known to the expert in thefield and are described in standard textbooks as Sambrook J. et al.,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989 and Ausubel F. et al.: CurrentProtocols in Molecular Biology 1987, J. Wiley and Sons, NY. There may bealso further purification steps before the nucleic acid detection stepis carried out as e.g. a precipitation step. The detection methods mayinclude but are not limited to the binding or intercalating of specificdyes as ethidium bromide which intercalates into the double-stranded DNAand changes its fluorescence thereafter. The purified nucleic acid mayalso be separated by electrophoretic methods optionally after arestriction digest and visualized thereafter. There are also probe-basedassays which exploit the oligonucleotide hybridization to specificsequences and subsequent detection of the hybrid. It is also possible tosequence the nucleic acid after further steps known to the expert in thefield. An example of a template-dependent nucleic acid polymerase is theZ05 DNA polymerase and mutations thereof. Other template-dependentnucleic acid polymerases useful in the invention are Taq polymerase andTth Polymerase. Yet other nucleic acid polymerases useful for themethods according to the invention are known to the skilled artisan.

In the context of the invention, the microbial nucleic acid and each ofthe control nucleic acids bind to different probes carrying differentlabels in order to render them distinguishable from one another duringdetection. Hence, said different probes can carry different fluorescentdyes, emitting fluorescent light at different wavelengths. Thesedifferent fluorescence signals can be advantageously detectedindependently from each other in different channels of a fluorescencedetector as used in most devices for performing real time PCR.

Also for example, results for the microbial nucleic acid, the firstcontrol nucleic acid and the second control nucleic acid are visualizedon a display in different masks, or in different displays.

In the sense of the invention, “purification”, “isolation” orextraction” of nucleic acids relate to the following: Before nucleicacids may be analyzed in one of the above-mentioned assays, they have tobe purified, isolated or extracted from biological samples containingcomplex mixtures of different components. Often, for the first steps,processes are used which allow the enrichment of the nucleic acids. Torelease the contents of cells or viral particles, they may be treatedwith enzymes or with chemicals to dissolve, degrade or denature thecellular walls or viral particles. This process is commonly referred toas lysis. The resulting solution containing such lysed material isreferred to as lysate. A problem often encountered during lysis is thatother enzymes degrading the component of interest, e.g.deoxyribonucleases or ribonucleases degrading nucleic acids, come intocontact with the component of interest during the lysis procedure. Thesedegrading enzymes may also be present outside the cells or may have beenspatially separated in different cellular compartments prior to lysis.As the lysis takes place, the component of interest becomes exposed tosaid degrading enzymes. Other components released during this processmay e.g. be endotoxins belonging to the family of lipopolysaccharideswhich are toxic to cells and can cause problems for products intended tobe used in human or animal therapy.

There is a variety of means to tackle the above-mentioned problem. It iscommon to use chaotropic agents such as guanidinium thiocyanate oranionic, cationic, zwitterionic or non-ionic detergents when nucleicacids are intended to be set free. It is also an advantage to useproteases which rapidly degrade the previously described enzymes orunwanted proteins. However, this may produce another problem as saidsubstances or enzymes can interfere with reagents or components insubsequent steps.

Enzymes which can be advantageously used in such lysis or samplepreparation processes mentioned above are enzymes which cleave the amidelinkages in protein substrates and which are classified as proteases, or(interchangeably) peptidases (see Walsh, 1979, Enzymatic ReactionMechanisms. W. H. Freeman and Company, San Francisco, Chapter 3).Proteases used in the prior art comprise alkaline proteases (WO98/04730) or acid proteases (U.S. Pat. No. 5,386,024). A protease whichhas been widely used for sample preparation in the isolation of nucleicacids in the prior art is proteinase K from Tritirachium album (see e.g.Sambrook J. et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1989) which is activearound neutral pH and belongs to a family of proteases known to theperson skilled in the art as subtilisins. Especially advantageous forthe use in lysis or sample preparation processes mentioned above is theenzyme esperase, a robust protease that retains its activity at bothhigh alkalinity and at high temperatures (EP 1 201 753).

In the sample preparation steps following the lysis step, the componentof interest is further enriched.

If the non-proteinaceous components of interest are e.g. nucleic acids,they are normally extracted from the complex lysis mixtures before theyare used in a probe-based assay.

There are several methods for the purification of nucleic acids, forexample:

-   -   sequence-dependent or biospecific methods as e.g.:    -   affinity chromatography    -   hybridization to immobilized capture oligonucleotides    -   sequence-independent or physico-chemical methods as e.g.:    -   liquid-liquid extraction with e.g. phenol-chloroform    -   precipitation with e.g. pure ethanol    -   extraction with filter paper    -   extraction with micelle-forming agents as        cetyl-trimethyl-ammonium-bromide    -   binding to immobilized, intercalating dyes, e.g. acridine        derivatives    -   adsorption to silica gel or diatomic earths    -   adsorption to magnetic glass particles (MGP) or organo-silane        particles under chaotropic conditions

Particularly interesting for purification purposes is the adsorption ofnucleic acids to a glass surface although other surfaces are possible.Many procedures for isolating nucleic acids from their naturalenvironment have been proposed in recent years by the use of theirbinding behavior to glass surfaces. If unmodified nucleic acids are thetarget, a direct binding of the nucleic acids to a material with asilica surface is preferred because, among other reasons, the nucleicacids do not have to be modified, and even native nucleic acids can bebound. These processes are described in detail by various documents. InVogelstein B. et al., Proc. Natl. Acad. USA 76 (1979) 615-9, forinstance, a procedure for binding nucleic acids from agarose gels in thepresence of sodium iodide to ground flint glass is proposed. Thepurification of plasmid DNA from bacteria on glass dust in the presenceof sodium perchlorate is described in Marko M. A. et al., Anal. Biochem.121 (1982) 382-387. In DE-A 37 34 442, the isolation of single-strandedM13 phage DNA on glass fiber filters by precipitating phage particlesusing acetic acid and lysis of the phage particles with perchlorate isdescribed. The nucleic acids bound to the glass fiber filters are washedand then eluted with a methanol-containing Tris/EDTA buffer. A similarprocedure for purifying DNA from lambda phages is described in Jakobi R.et al., Anal. Biochem. 175 (1988) 196-201. The procedure entails theselective binding of nucleic acids to glass surfaces in chaotropic saltsolutions and separating the nucleic acids from contaminants such asagarose, proteins or cell residue. To separate the glass particles fromthe contaminants, the particles may be either centrifuged or fluids aredrawn through glass fiber filters. This is a limiting step, however,that prevents the procedure from being used to process large quantitiesof samples. The use of magnetic particles to immobilize nucleic acidsafter precipitation by adding salt and ethanol is more advantageous anddescribed e.g. in Alderton R. P. et al., S., Anal. Biochem. 201 (1992)166-169 and PCT GB 91/00212. In this procedure, the nucleic acids areagglutinated along with the magnetic particles. The agglutinate isseparated from the original solvent by applying a magnetic field andperforming a wash step. After one wash step, the nucleic acids aredissolved in a Tris buffer. This procedure has a disadvantage, however,in that the precipitation is not selective for nucleic acids. Rather, avariety of solid and dissolved substances are agglutinated as well. As aresult, this procedure cannot be used to remove significant quantitiesof any inhibitors of specific enzymatic reactions that may be present.Magnetic, porous glass is also available on the market that containsmagnetic particles in a porous, particular glass matrix and is coveredwith a layer containing streptavidin. This product can be used toisolate biological materials, e.g., proteins or nucleic acids, if theyare modified in a complex preparation step so that they bind covalentlyto biotin. Magnetizable particular adsorbents proved to be veryefficient and suitable for automatic sample preparation. Ferrimagneticand ferromagnetic as well as superparamagnetic, pigments are used forthis purpose. MGPs and methods using magnetic glass particles are, forexample, those described in WO 01/37291. Particularly useful for thenucleic acid isolation in the context of the invention is the methodaccording to R. Boom et al. (J Clin Microbiol. 28 (1990), 495-503).

After the purification or isolation of the nucleic acids including thetarget nucleic acid from their natural surroundings, the target nucleicacid may be detected.

In an embodiment, the method of the invention includes steps to avoidcontamination. For example, an enzymatic method utilizing uracil-DNAglycosylase is described in U.S. Pat. Nos. 5,035,996, 5,683,896 and5,945,313 to reduce or eliminate contamination between one thermocyclerrun and the next. In addition, standard laboratory containment practicesand procedures are desirable when performing the method of theinvention. Containment practices and procedures include, but are notlimited to, separate work areas for different steps of a method,containment hoods, barrier filter pipette tips and dedicated airdisplacement pipettes. Consistent containment practices and proceduresby personnel are necessary for accuracy in a diagnostic laboratoryhandling clinical samples.

The methods set out above can be based on Fluorescence Resonance EnergyTransfer (FRET) between a donor fluorescent moiety and an acceptorfluorescent moiety. A representative donor fluorescent moiety isfluorescein, and representative corresponding acceptor fluorescentmoieties include LC-Red 640, LC-Red 705, Cy5, and Cy5.5. Typically, thedetecting step includes exciting the sample at a wavelength absorbed bythe donor fluorescent moiety and visualizing and/or measuring thewavelength emitted by the corresponding acceptor fluorescent moiety.According to the invention, the detection is followed by quantitatingthe FRET. For example, the detecting step is performed after eachcycling step. For example, the detecting step is performed in real time.By using commercially available real-time PCR instrumentation (e.g.,LightCycler™ or TaqMan®), PCR amplification and detection of theamplification product can be combined in a single closed reactioncompartment such as, e.g., a cuvette with dramatically reduced cyclingtime. Since detection occurs concurrently with amplification, thereal-time PCR methods obviate the need for manipulation of theamplification product, and diminish the risk of cross-contaminationbetween amplification products. Real-time PCR greatly reducesturn-around time and is an attractive alternative to conventional PCRtechniques in the clinical laboratory.

In example, the following patent applications describe real-time PCR asused in the LightCycler™ technology: WO 97/46707, WO 97/46714 and WO97/46712. The LightCycler™ instrument is a rapid thermal cycler combinedwith a microvolume fluorometer utilizing high quality optics. This rapidthermocycling technique uses thin glass cuvettes as reaction vessels.Heating and cooling of the reaction chamber are controlled byalternating heated and ambient air. Due to the low mass of air and thehigh ratio of surface area to volume of the cuvettes, very rapidtemperature exchange rates can be achieved within the thermal chamber.

TaqMan® technology utilizes a single-stranded hybridization probelabeled with two fluorescent moieties. When a first fluorescent moietyis excited with light of a suitable wavelength, the absorbed energy istransferred to a second fluorescent moiety according to the principlesof FRET. The second fluorescent moiety is generally a quencher molecule.Typical fluorescent dyes used in this format are for example, amongothers, FAM, HEX, CY5, JA270, Cyan and CY5.5. During the annealing stepof the PCR reaction, the labeled hybridization probe binds to the targetnucleic acid (i.e., the amplification product) and is degraded by the 5′to 3′ exonuclease activity of the Taq or another suitable polymerase asknown by the skilled artisan, such as the Z05 polymerase, during thesubsequent elongation phase. As a result, the excited fluorescent moietyand the quencher moiety become spatially separated from one another. Asa consequence, upon excitation of the first fluorescent moiety in theabsence of the quencher, the fluorescence emission from the firstfluorescent moiety can be detected.

In both detection formats described above, the intensity of the emittedsignal can be correlated with the number of original target nucleic acidmolecules.

As an alternative to FRET, an amplification product can be detectedusing a double-stranded DNA binding dye such as a fluorescent DNAbinding dye (e.g., SYBRGREEN I® or SYBRGOLD® (Molecular Probes)). Uponinteraction with the double-stranded nucleic acid, such fluorescent DNAbinding dyes emit a fluorescence signal after excitation with light at asuitable wavelength. A double-stranded DNA binding dye such as a nucleicacid intercalating dye also can be used. When double-stranded DNAbinding dyes are used, a melting curve analysis is usually performed forconfirmation of the presence of the amplification product.

Molecular beacons in conjunction with FRET can also be used to detectthe presence of an amplification product using the real-time. PCRmethods of the invention. Molecular beacon technology uses ahybridization probe labeled with a first fluorescent moiety and a secondfluorescent moiety. The second fluorescent moiety is generally aquencher, and the fluorescent labels are typically located at each endof the probe. Molecular beacon technology uses a probe oligonucleotidehaving sequences that permit secondary structure formation (e.g., ahairpin). As a result of secondary structure formation within the probe,both fluorescent moieties are in spatial proximity when the probe is insolution. After hybridization to the amplification products, thesecondary structure of the probe is disrupted and the fluorescentmoieties become separated from one another such that after excitationwith light of a suitable wavelength, the emission of the firstfluorescent moiety can be detected.

Thus, in a method according to the invention is the method describedabove using FRET, wherein said probes comprise a nucleic acid sequencethat permits spatial proximity between said first and second fluorescentmoiety.

Efficient FRET can only take place when the fluorescent moieties are indirect local proximity and when the emission spectrum of the donorfluorescent moiety overlaps with the absorption spectrum of the acceptorfluorescent moiety.

Thus, in an embodiment of the invention, said donor and acceptorfluorescent moieties are within no more than 5 nucleotides of each otheron said probe.

In a further embodiment, said acceptor fluorescent moiety is a quencher.

As described above, in the TaqMan format, during the annealing step ofthe PCR reaction, the labeled hybridization probe binds to the targetnucleic acid (i.e., the amplification product) and is degraded by the 5′to 3′ exonuclease activity of the Taq or another suitable polymerase asknown by the skilled artisan, such as the Z05 polymerase, during thesubsequent elongation phase.

Thus, in an embodiment, in the method according to the invention,amplification employs a polymerase enzyme having 5′ to 3′ exonucleaseactivity.

The method according to the invention can be advantageously applied onviral nucleic acids. Thus, in an embodiment of the invention, themicrobial nucleic acid is a viral nucleic acid.

Among viral nucleic acids, the method according to the invention can beadvantageously be applied on HCV. Thus, in an embodiment of theinvention, the microbial nucleic acid is a nucleic acid of HCV. However,it must be understood that the invention can also be applied on anyother microbial nucleic acid.

An example how to perform calculation of quantitative results in theTaqMan format based on an internal standard is described in thefollowing. A titer is calculated from input data of instrument-correctedfluorescence values from an entire PCR run. A set of samples containinga target nucleic acid such as a microbial nucleic acid and a firstcontrol nucleic acid serving as an internal quantitative standardnucleic acid undergo PCR on a thermocycler using a temperature profileas specified. At selected temperatures and times during the PCR profilesamples are illuminated by filtered light and the filtered fluorescencedata are collected for each sample for the target nucleic acid and theinternal quantitative standard nucleic acid. After a PCR run iscomplete, the fluorescence readings are processed to yield one set ofdye concentration data for the internal quantitative standard nucleicacid and one set of dye concentration data for the target nucleic acid.Each set of dye concentration data is processed in the same manner.After several plausibility checks, the elbow values (CT) are calculatedfor the internal quantitative standard nucleic acid and the targetnucleic acid. The elbow value is defined as the point where thefluorescence of the target nucleic acid or the internal quantitativestandard nucleic acid crosses a predefined threshold (fluorescenceconcentration). Titer determination is based on the assumptions that thetarget nucleic acid and the internal quantitative standard nucleic acidare amplified with the same efficiency and that at the calculated elbowvalue equal amounts of amplicon copies of target nucleic acid andquantitative standard nucleic acid are amplified and detected.Therefore, the (CTQS−CTtarget) is linear to log (target conc/QS conc),wherein “QS” stands for the internal quantitative standard nucleic acid.The titer T can then be calculated for instance by using a polynomialcalibration formula as in the following equation:

T′=10(a(CTQS−CTtarget)2+b(CTQS−CTtarget)+c)

The polynomial constants and the concentration of the internalquantitative standard nucleic acid are known, therefore the onlyvariable in the equation is the difference (CTQS−CTtarget).

An example how to perform calculation of qualitative results in theTaqMan is described in the following: Input data of instrument-correctedfluorescence values from an entire PCR run are analyzed. A set ofsamples possibly containing a target nucleic acid such as a microbialnucleic acid and a control nucleic acid serving as a qualitativeinternal control nucleic acid undergo PCR on a thermocycler using atemperature profile as specified. At selected temperatures and timesduring the PCR profile samples are illuminated by filtered light and thefiltered fluorescence data are collected for each sample for the targetnucleic acid and the qualitative internal control nucleic acid. After aPCR run is complete, the fluorescence readings are processed to yieldone set of dye concentration data for the qualitative internal controlnucleic acid and one set of dye concentration data for the targetnucleic acid. Each set of dye concentration data is processed in thesame manner. The elbow values (CT) are calculated for the qualitativeinternal control nucleic acid and, if present, the target nucleic acid.The elbow value is defined as the point where the fluorescence of thetarget nucleic acid or the internal quantitative standard nucleic acidcrosses a predefined threshold (fluorescence concentration). Thequalitative internal control is valid if a CT within specified ranges isobtained and if the fluorescence rises above a predefined minimumfluorescence intensity. If no target CT is obtained, the qualitativeinternal control must be valid. If a target CT is obtained indicatingpresence of target nucleic acid in the sample, the qualitative internalcontrol can be valid or invalid.

Embodiments of the invention are any of the methods described above,wherein said first and said second control nucleic acid are providedwithin one control reagent.

The concept of a control reagent with a first and a second controlnucleic acid in different concentrations for reliable qualitativedetection and at the same time for reliable quantification can beadvantageously used for any respective assay targeting a microbialnucleic acid.

Thus, another aspect of the invention is the use of a first and a secondcontrol nucleic acid in different concentrations for simultaneouslydetecting and quantifying a microbial nucleic acid by real time PCR.Further for example is the use described above, wherein said first andsecond control nucleic acid are amplified by the same primer pair ordifferent primer pairs but hybridize to different probes.

Particularly favorable is a use of this concept in the method accordingto the invention. Thus, in another aspect, the invention concerns theuse of a first and a second control nucleic acid in differentconcentrations for simultaneously detecting and quantifying a microbialnucleic acid according to the method or methods described above.

The invention also provides a kit for simultaneously detecting andquantifying a microbial nucleic acid in a biological sample by real timePCR, said kit comprising a first and a second control nucleic acid indifferent concentrations, one or more primer pairs specificallyhybridizing to distinct sequence portions of said microbial nucleic acidand to distinct sequence portions of said first and second controlnucleic acid, and probes specifically hybridizing to each of thesequences amplified by said one or more primer pairs, wherein said firstand second control nucleic acids are provided within one controlreagent.

In an embodiment, the invention provides a kit for simultaneouslydetecting and quantifying a microbial nucleic acid in a biologicalsample according to any of the methods described supra, said kitcomprising a first and a second control nucleic acid in differentconcentrations, one or more primer pairs specifically hybridizing todistinct sequence portions of said microbial nucleic acid and todistinct sequence portions of said first and second control nucleicacid, and probes specifically hybridizing to each of the sequencesamplified by said one or more primer pairs.

A further aspect of the invention is a kit as described above, whereinsaid first and second control nucleic acid are amplified by the sameprimer pair but hybridize to different probes, thus providing for acompetitive setup. However, it has to be understood that non- orpartially competitive assays and kits for carrying out the respectivemethods are also comprised by the invention.

Such kits may further comprise, as known in the art, plastics ware whichcan be used during the sample preparation procedure as e.g. microtiterplates in the 96 or 384 well format or ordinary reaction tubesmanufactured e.g. by Eppendorf, Hamburg, Germany and all other reagentsfor carrying out the method according to the invention. Therefore, thekit can additionally contain a material with an affinity to nucleicacids, for example the material with an affinity to nucleic acidscomprises a material with a silica surface. Further for example, thematerial with a silica surface is a glass. For example, the materialwith an affinity to nucleic acids is a composition comprising magneticglass particles. The kit can further or additionally comprise a proteasereagent and a lysis buffer containing e.g. chaotropic agents, detergentsor alcohols or mixtures thereof allowing for the lysis of cells. Thesecomponents of the kit according to the invention may be providedseparately in tubes or storage containers. Depending on the nature ofthe components, these may be even provided in a single tube or storagecontainer. The kit may further or additionally comprise a washingsolution which is suitable for the washing step of the magnetic glassparticles when a nucleic acid is bound thereto. This washing solutionmay contain ethanol and/or chaotropic agents in a buffered solution orsolutions with an acidic pH without ethanol and/or chaotropic agents asdescribed above. Often the washing solution or other solutions areprovided as stock solutions which have to be diluted before use. The kitmay further or additionally comprise an eluent or elution buffer, i.e. asolution or a buffer (e.g. 10 mM Tris, 1 mM EDTA, pH 8.0) or pure waterto elute the nucleic acid bound to the magnetic glass particles.Further, additional reagents or buffered solutions may be present whichcan be used for the purification process of a nucleic acid.

In another embodiment, the kit contains a polymerase enzyme having 5′ to3′ exonuclease activity. Another embodiment is that the kit contains anenzyme with reverse transcriptase activity.

In another embodiment, the kit contains a polymerase enzyme having 5′ to3′ exonuclease activity and reverse transcriptase activity.

In an embodiment of the invention, the method according to the inventionis embedded in a sequence of methods carried out within an analyticalsystem, thereby forming an automatable process.

Therefore, an aspect of the invention is the following:

An analytical system for simultaneously detecting and quantifying amicrobial nucleic acid in a biological sample by real time PCR, saidsystem comprising

-   -   a sample preparation module comprising a lysis buffer and a        vessel for isolating and purifying said microbial nucleic acid    -   an amplification and detection module comprising a reaction        receptacle in which the method described above is performed    -   a kit as described above.

The sample preparation module can advantageously comprise components forthe sample preparation procedures described supra, i.e. for examplemagnetic glass particles and a magnet for separating them from thesolution, a protease reagent, chaotropic salt solutions and one or morevessels that contain the crude sample and the reagents required forsample preparation.

The reaction receptacle in the amplification and detection module canfor example be a microtiter plate, a centrifugation vial, a lysis tube,or any other type of vessel suitable for containing a reaction mixtureaccording to the invention.

In an embodiment of the invention, the analytical system contains astorage module containing the reagents for performing the method of theinvention.

Said storage module can further contain other components useful for themethod of the invention, e.g. disposables such as pipet tips or evenvessels to be used as reaction receptacles within the amplification anddetection module.

In an embodiment of the invention, the analytical system includes apreanalytical system module for transferring the biological sample fromprimary tubes to vessels usable on the analytical system.

In yet another embodiment of the invention, the analytical systemcontains a transfer module for transferring the biological sample fromthe sample preparation module to the amplification and detection module.

Even though it is possible to carry out said transfer manually, it ispreferable to use an automated system wherein the transfer is performede.g. by a robotic device such e.g. as a motor-driven mobile rack orrobotic pivot arm.

Automatable process means that the steps of the process are suitable tobe carried out with an apparatus or machine capable of operating withlittle or no external control or influence by a human being. Automatedmethod means that the steps of the automatable method are carried outwith an apparatus or machine capable of operating with little or noexternal control or influence by a human being. Only the preparationsteps for the method may have to be done by hand, e.g. storagecontainers have to be filled and put into place, the choice of sampleshas to be performed by a human being and further steps known to theexpert in the field, e.g. the operation of a controlling computer. Theapparatus or machine may e.g. automatically add liquids, mix the samplesor carry out incubation steps at specific temperatures. Typically, sucha machine or apparatus is a robot controlled by a computer which carriesout a program in which the single steps and commands are specified.

Thus, for example, the analytical system according to the inventionfurther comprises a control unit for controlling system components.

Such a control unit may comprise a software for ensuring that thedifferent components of said analytical system work and interactcorrectly and with the correct timing, e.g. moving components such asthe sample to the reaction module in a coordinated manner. The controlunit may also comprise a processor running a real-time operating system(RTOS), which is a multitasking operating system intended for real-timeapplications. In other words the system processor is capable of managingreal-time constraints, i.e. operational deadlines from event to systemresponse regardless of system load. It controls in real time thatdifferent units within the system operate and respond correctlyaccording to given instructions.

All other embodiments and specific descriptions of embodiments of theuses, kits and analytical systems according to the invention are thosementioned for the method according to the invention.

DESCRIPTION OF THE FIGURES

FIG. 1 a:

Internal control growth curves for quantitative real time PCR reactionsof the commercial COBAS AmpliPrep/COBAS TaqMan HIV-1 test are shown inthe graph. The PCR reactions contained HIV-1 target concentrationsspanning a 5 log 10 range and a target negative sample. If high targetconcentrations are present in the reaction (see “Target high positive”)the fluorescence level which is reached by the internal quantitativestandard nucleic acid (QS) is significantly lower than in the case ofPCR reactions with no HIV-1 target present (see “Target negative”). Asthe QS must be valid at all target concentrations in order to be able tocalculate a titer for a quantitative result output, the minimalfluorescence intensity level setting (“RFImin”) for the QS is low.RFImin settings for a quantitative result output in the three commercialtests COBAS AmpliPrep/COBAS TaqMan HBV, HCV and HIV-1 are given in Table1.

FIG. 1 b:

Internal control growth curves for real time PCR reactions of thecommercial COBAS AmpliPrep/COBAS TaqMan HIV-1 test are shown in thegraph. All PCR reactions contained HIV-1 target negative samples. Thefluorescence level of the internal control growth curves is bundledwithin a narrow range and the minimal fluorescence intensity levelsetting (“RFImin”) for the qualitative internal control nucleic acid(IC) is high. In target positive reactions the IC may fall below theRFImin and may become invalid; in the presence of target the result forthe PCR reaction is still valid. RFImin settings as optimized for aqualitative result output for the tests COBAS AmpliPrep/COBAS TaqManHBV, HCV and HIV-1 are given in Table 1.

FIG. 2:

Production of armored RNA particles: expression vector for theproduction of armored RNA particles and electron microscopy of armoredRNA particles. Armored RNA particles are composed in E. coli. Afterinduction of lac-Operon mRNA is transcribed that includes the MS-2bacteriophage gene of MS-2 coat protein, the control sequence, apackaging signal and a the plasmid sequence upstream the TrrnBterminator. After the coat protein is translated, composition of theparticle occurs spontaneously packing one copy of mRNA.

FIG. 3:

Normalized growth curves obtained for a real time PCR reactioncontaining HCV target, a first control nucleic acid and a second controlnucleic acid.

FIG. 4 a:

Simultaneous amplification of HCV-RNA and QS. The fluorescence intensityof both QS and HCV-RNA are lower than in a reaction leading to standardsignals (see FIG. 4 b).

FIG. 4 b:

Simultaneous amplification of HCV-RNA and QS. Both reactions result instandard fluorescence intensities. For comparison see FIG. 4 a withsuppressed fluorescence curves, also note the different magnitude in thetwo figures.

EXAMPLES

The following examples are offered to illustrate, but not to limit theclaimed invention.

Example 1 A First Control Nucleic Acid is Evaluated According toDifferent Criteria for Obtaining a Qualitative and a Quantitative ResultOutput

The COBAS AmpliPrep/COBAS TaqMan HBV, HCV and HIV-1 tests arecommercially available real-time PCR tests which have been optimized foraccurate quantification of the viral targets HBV, HCV and HIV-1. Theassays are used on either the fully automated COBAS AmpliPrep/COBASTaqMan system including a docking station for the automated transfer ofthe PCR reaction mix from the sample preparation unit to theamplification/detection unit or on the COBAS AmpliPrep/COBAS TaqMansystem with manual transfer or on the COBAS AmpliPrep/COBAS TaqMan 48system.

The COBAS AmpliPrep/COBAS TaqMan HCV test was used as an example toinvestigate the approach to provide simultaneous quantitative andqualitative test results with one set of identical reagents. The testhas a Limit of Detection of 15 IU/mL and shows state of the artsensitivity, comparable or better to quantitative but also qualitativeHCV tests on the market. Due to this high sensitivity there have beenattempts to also use the test as qualitative HCV test. The main focus ofa quantitative test is to provide accurate HCV titers whereas the mainfocus of a qualitative test is to ensure high sensitivity of the assay.As the COBAS AmpliPrep/COBAS TaqMan HCV test originally was developedfor quantitative monitoring of treatment success an analysis wasconducted whether the assay can also be used without any changes toprovide reliable qualitative results. A review of data revealed that inthe rare event of a less efficient PCR the sensitivity of 15 IU/mL isnot guaranteed by the quantitative test with its current data analysissettings. An example of a PCR reaction which is inhibited but shows avalid QS result and a failed detection of target (2183 IU/mL) is givenbelow:

An inhibited PCR reaction which yielded a valid result due to a valid QSand a result of “Target not detected” is shown in FIG. 4 a. The QS wasvalid because it exceeded the fluorescence threshold settings whichcorresponded to 0.8 relative fluorescence units.

The repeat testing of the same sample showed a standard PCR reaction anda HCV titer result of 2183 IU/mL (FIG. 4 b).

Thus the quantitative COBAS AmpliPrep/COBAS TaqMan HCV test without anychanges cannot be used as qualitative test.

Using the above mentioned test the data analysis parameters settings ofthe IC/QS control nucleic acid were optimized such that a reliablequalitative result output is achieved. In order to demonstrate thegeneral applicability, the optimization was additionally undertaken forall three commercial HBV, HCV and HIV-1 assays on the COBASAmpliPrep/COBAS TaqMan system. By significantly raising the minimalrelative fluorescence intensity (RFImin) threshold in the parametersettings the sensitivity of all three quantitative tests is ensured suchthat they can be used to provide a reliable qualitative result output.The relevant parameter settings are shown in Table 1 below:

TABLE 1 RFImin settings for the internal control of three COBASAmpliPrep/COBAS TaqMan tests in the commercially available quantitativeapplication and in a qualitative application. Quantitative QualitativeRFImin IQS RFImin IQS HBV test on the COBAS 1.5 10.4 AmpliPrep/COBASTaqman HCV test on the COBAS 1.8 9.0 AmpliPrep/COBAS Taqman HIV-1 teston the COBAS 1.2 20.4 AmpliPrep/COBAS Taqman

The qualitative RFImin settings of 9.0 would invalidate the inhibitedPCR reaction in the example given for HCV above.

The above RFImin settings for the qualitative result output cannot beused simultaneously for the quantitative result output because thepresence of target may affect the fluorescence intensity of the IC/QSgrowth curves as shown in FIG. 1 a. As a result the qualitative RFIminsettings lead to a high percentage of IC/QS invalid reactions in thepresence of high target concentrations. In contrast, this does notaffect the validity of the qualitative result output as the IC must notbe valid in the presence of target. The validity of the quantitativereaction is significantly affected, though.

Thus, if the same test reagents with one IC/QS are to be used to provideboth a reliable qualitative result output with guaranteed sensitivity ofthe test and a reliable quantitative test result with acceptably lowlevels of QS invalid results this can only be achieved by analyzing theraw data with two different sets of parameter settings as suggestedhere.

Example 2 A First and a Second Control Nucleic Acid in DifferentConcentrations are Used for Obtaining a Quantitative and a QualitativeResult Output with the Same Test Reagents

The goal is to provide both a reliable qualitative result output and areliable quantitative test result with the same set of test reagents. Inthis approach to address this goal the internal control nucleic acidcontaining reagent comprises a formulation with two different armoredRNA particles. The following reagents can be used for this experiment:

a) Sample preparation reagents of the COBAS AmpliPrep/COBAS TaqMan HCVtest (magnetic particle suspension; protease solution, lysis buffer;elution buffer)

b) QS armored particle at a concentration level of about 1000copies/reaction and an IC armored particle at a concentration level ofabout 100 copies/reaction. The production of armored RNA particles isshown in FIG. 2 below. The transcripts enclosed by the armored particleshave different primer binding sites and different probe binding sites.The probes are labelled with the fluorescent labels HEX and CY5 togetherwith the black hole quencher BHQ.

c) Magnesium reagent

d) Mastermix reagent comprising:

-   -   Z05 DNA polymerase and UNG    -   three different primer pairs for the target HCV, for the first        and for the second control nucleic acid,    -   three different probes for the target labeled with FAM and BHQ,        for the first control nucleic acid labeled with HEX and BHQ and        for the second control nucleic acid labeled with CY5 and BHQ    -   Aptamer    -   dNTPs (dUTP, dATP, dCTP, dGTP, dTTP)    -   mastermix buffer ingredients (potassium acetate, glycerol,        Tricine, DMSO, Betaine, IGEPAL, water),

The sample preparation and the amplification/detection steps areperformed using the specimen extraction parameters and the PCR fileestablished for the COBAS AmpliPrep/COBAS TaqMan HCV Test. Growth curvesfor the three different fluorescent dyes are obtained and are presentedin FIG. 3.

For the HCV samples the titers are determined as follows to obtain thequantitative result output: after a PCR run is complete, thefluorescence readings are processed to yield one set of dyeconcentration data for the QS nucleic acid (HEX fluorescent label), oneset of dye concentration data for the qualitative standard nucleic acid(CY5 fluorescent label), and one set of dye concentration data for thetarget nucleic acid (FAM fluorescent label). All three sets of dyeconcentration data are processed in the same manner. The elbow values(CT) are calculated for the quantitative and the qualitative standardnucleic acid as well as the target nucleic acid. The elbow value isdefined as the point where the fluorescence of the target nucleic acidor the two internal control nucleic acids crosses a predefined threshold(fluorescence concentration).

For the quantitative result output titer determination is done byanalyzing only the dye concentration data for HEX and FAM. Titerdetermination is based on the assumption that the target nucleic acidand the QS nucleic acid are amplified with the same efficiency and thatat the calculated elbow value equal amounts of amplicon copies of targetnucleic acid and QS nucleic acid are amplified and detected. Therefore,the (CTQS−CTtarget) is linear to log (target conc/QS conc). The titer Tcan then be calculated for instance by using a polynomial calibrationformula as in the following equation in which the only variable in theequation is the difference (CTQS−CTtarget):

T′=10(a(CTQS−CTtarget)2+b(CTQS−CTtarget)+c)

For the qualitative result output only the dye concentration data forCY5 and FAM are analyzed. By comparing the dye concentration data forCY5 and FAM the software determines whether the PCR reaction is targetnegative or positive. If the PCR reaction is target positive the ICresult is neglected and thus may be valid or invalid. If the PCRreaction is target negative, the result is only valid if the IC shows avalid result. In the example shown above, all reactions contain lowlevels of HCV target and—although not relevant for the validity of therespective test results—all reactions show a valid IC.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, sequence accessionnumbers, patents, and patent applications cited herein are herebyincorporated by reference in their entirety for all purposes.

1. A method for simultaneously detecting and quantifying a microbialnucleic acid in a biological sample, the method comprising: a) providinga reaction mixture comprising: a first control nucleic acid, one or moreprimer pairs that hybridize to distinct sequence portions of themicrobial nucleic acid and to distinct sequence portions of the firstcontrol nucleic acid, and two or more probes that hybridize to each ofthe sequences amplified by the one or more primer pairs, wherein themicrobial nucleic acid and the first control nucleic acid hybridize todifferent probes; b) adding the biological sample to the reactionmixture; c) performing one or more cycling steps, wherein each cyclingstep comprises: an amplifying step comprising producing one or moreamplification products derived from the microbial nucleic acid ifpresent in the sample and producing an amplification product derivedfrom the first control nucleic acid, and a hybridizing step comprisinghybridizing the amplification products with the two or more probes,wherein the two or more probes are each labeled with a donor fluorescentmoiety and a corresponding acceptor fluorescent moiety and each of thetwo or more probes carries a different fluorescent moiety dye; and d)detecting and measuring fluorescent signals generated in step c),wherein the fluorescent signals generated by the first control nucleicacid and the microbial nucleic acid are proportional to theirconcentration and are indicative of the detection and quantification ofthe microbial nucleic acid, and wherein the fluorescent signals from thefirst control nucleic acid are indicative of an amplification occurringin the amplifying step even in the absence of the fluorescent signalsfrom the microbial nucleic acid.
 2. The method of claim 1, wherein thefluorescent signals from the first control nucleic acid are analyzed bydifferent criteria to obtain a quantitative result and/or a qualitativeresult.
 3. A method for simultaneously detecting and quantifying amicrobial nucleic acid in a biological sample, said method comprising:a) providing a reaction mixture comprising: a first and a second controlnucleic acid in different concentrations, one or more primer pairs thathybridize to distinct sequence portions of the microbial nucleic acidand to distinct sequence portions of the first and the second controlnucleic acid, and three or more probes that hybridize to each of thesequences amplified by the one or more primer pairs, wherein themicrobial nucleic acid and the first and second control nucleic acideach hybridize to different probes; b) adding the biological sample tothe reaction mixture; c) performing one or more cycling steps, whereineach cycling step comprises: an amplifying step comprising producing oneor more amplification products derived from the microbial nucleic acidif present in the sample and producing an amplification product derivedfrom the first control nucleic acid and an amplification product derivedfrom the second control nucleic acid, and a hybridizing step comprisinghybridizing the amplification products with the three or more probes,wherein the three or more probes are each labeled with a donorfluorescent moiety and a corresponding acceptor fluorescent moiety andeach of the three or more probes carries a different fluorescent moietydye; and d) detecting and measuring fluorescent signals generated instep c), wherein the fluorescent signals generated by the first controlnucleic acid and the microbial nucleic acid are proportional to theirconcentration and are indicative of the detection and quantification ofthe microbial nucleic acid, and wherein the fluorescent signals from thesecond control nucleic acid are indicative of an amplification occurringin the amplifying step even in the absence of the fluorescent signalsfrom the microbial nucleic acid.
 4. The method of claim 3, wherein thefirst control nucleic acid is a quantitative standard nucleic acid andthe second control nucleic acid is a qualitative internal controlnucleic acid.
 5. The method of claim 1, further comprising: in step d)determining the quantity of the microbial nucleic acid in the biologicalsample by comparison of the signals generated by the microbial nucleicacid and the first control nucleic acid.
 6. The method of claim 1,wherein the amplifying step employs a polymerase enzyme having 5′ to 3′exonuclease activity.
 7. The method of claim 3, wherein the firstcontrol nucleic acid is present in a concentration of 20-5000 times thelimit of detection of the microbial nucleic acid, and wherein the secondcontrol nucleic acid is present in a concentration of 1-25 times thelimit of detection of the microbial nucleic acid.
 8. The method of claim3, wherein the first and the second control nucleic acid are providedwithin one control reagent.
 9. A kit for simultaneously detecting andquantifying a microbial nucleic acid in a biological sample, the kitcomprising a first and a second control nucleic acid in differentconcentrations, one or more primer pairs that hybridize to distinctsequence portions of the microbial nucleic acid and to distinct sequenceportions of the first and second control nucleic acid, and probes thathybridize to each of the sequences amplified by the one or more primerpairs, wherein the first and second control nucleic acids are providedwithin one control reagent.
 10. A kit for simultaneously detecting andquantifying a microbial nucleic acid in a biological sample according tothe method of claim 3, the kit comprising a first and a second controlnucleic acid in different concentrations, one or more primer pairs thathybridize to distinct sequence portions of the microbial nucleic acidand to distinct sequence portions of the first and second controlnucleic acid, and probes that hybridize to each of the sequencesamplified by the one or more primer pairs.
 11. The kit of the claim 10,wherein the first and second control nucleic acid are amplified by thesame primer pair but hybridize to different probes.
 12. An analyticalsystem for simultaneously detecting and quantifying a microbial nucleicacid in a biological sample, the system comprising: a sample preparationmodule comprising a lysis buffer and a vessel for isolating andpurifying the microbial nucleic acid, and an amplification and detectionmodule comprising a reaction receptacle in which the method according toclaim 1 or 3 is performed.
 13. The analytical system of claim 12additionally comprising a transfer module for transferring thebiological sample from the sample preparation module to the reactionreceptacle.